The overall goal of this proposal is to understand the role macroH2A histone variants play in transcriptional regulation in cancer and senescence. MacroH2A is a histone variant found in heterochromatic regions of the genome, which are typically associated with gene repression. However, macroH2A does not generally play a causative role in heterochromatin-associated repression of transcription. Instead, macroH2A is required for a subset of its targets to be expressed from within a heterochromatic environment. The human genome encodes three macroH2A variants. MacroH2A1.1 and macroH2A1.2 are alternative splice products of the H2AFY gene, while macroH2A2 is encoded by H2AFY2. These variants are functionally distinct; while macroH2A1.1 interacts with signaling molecules such as poly(ADP-ribose) (PAR) generated by NAD+utilizing enzyme PARP-1, macroH2A1.2 and macroH2A2 cannot. Recently, changes in alternative splicing of H2AFY which reduce macroH2A1.1 expression have been associated with several cancer types and have even been shown to predict relapse after treatment. MacroH2A has also been implicated in an important tumor suppressive mechanism, cellular senescence, making the link between this histone variant and cancer even more compelling. As suggested by these observations, our broad hypothesis is that macroH2A1.1 has functions in regulating gene expression that are distinct from other macroH2A variants ultimately playing a role in tumor suppression. In order to test this hypothesis and to elucidate the distinct roles of each macroH2A variant, three specific aims are proposed. The first aim is to determine the role of macroH2A variants on gene expression in cancer and cellular senescence. This goal will be achieved by using genome-wide approaches to determine the pattern of genomic localization for each of the histone variants. In addition, we will perform expression analysis under conditions where we modulate the expression of macroH2A variants. The second aim is to determine the mechanisms that regulate macroH2A1 splicing in normal and cancer cells. To achieve this goal we will use both bioinformatics and a minigene splicing assay to determine both the cis- and trans-acting elements that contribute to the regulation of H2AFY alternative splicing. The third aim is to determine the role of PARP activity in macroH2A1.1-specific target gene expression. PARP-1 interacts with macroH2A1.1 through PAR binding, suggesting that macroH2A1.1 and PARP-1 may functionally collaborate to regulate gene expression. This hypothesis will be tested using both pharmacological inhibition and RNAi to modulate the level of PARP-1 activity, and assess the effect on factor recruitment to and transcriptional activity of macroH2A1.1 target genes. PARP-1 is a current therapeutic target in cancer treatment, highlighting the need to understand the functional connection between these two cancer-relevant molecules.